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Abstract:

A Pseudomonas sp. strain TKU015 is deposited under DSMZ GmbH (Deutsche
Sammlung von Mikroorganismen und Zellkulturen GmbH) Number DSM 21747).
The Pseudomonas sp. strain TKU015 can be used to produce chitinase,
chitosanase and nattokinase. A method of producing chitinase, chitosanase
and nattokinases can use the Pseudomonas sp. strain TKU015.

Claims:

1. A Pseudomonas sp. strain TKU015, which is deposited under DSMZ GmbH
(Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) Number DSM
21747), and can be used to produce chitinase, chitosanase and
nattokinase.

2. The Pseudomonas sp. strain TKU015 as claimed in claim 1, wherein the
strain is selected from soil and cultured using shrimp shell powder as
unique carbon/nitrogen source.

3. The Pseudomonas sp. strain TKU015 as claimed in claim 2, wherein the
soil is dissolved in germfree water thereby obtaining a solution, the
solution is coated on a solid culture medium comprising 0.2-1.5% by
weight of shrimp shell powder (SSP), 0.05-0.2% by weight of
K2HPO4, 0.02-0.1% by weight of MgSO.sub.4.7H20 and 0.5-3%
by weight of agar, the culture medium is cultured a predetermined period
thereby obtaining the Pseudomonas sp. strain TKU015.

5. The supernatant as claimed in claim 4, wherein the culture medium is
liquid medium comprised of 0.2-1.5% by weight of shrimp shell powder
(SSP), 0.05-0.2% by weight of K2HPO4, and 0.02-0.1% by weight
of MgSO.sub.4.7H2O, Pseudomonas sp. strain TKU015 is cultured in the
liquid medium using a shaking culture method, and the supernatant is
centrifugally separated from the liquid cultured medium.

6. The supernatant as claimed in claim 5, wherein the liquid medium is
comprised of 0.5% by weight of shrimp shell powder (SSP), 0.1% by weight
of K2HPO4, and 0.05% by weight of MgSO.sub.4.7H2O, pH
value of the liquid medium is 8, Pseudomonas sp. strain TKU015 is
cultured in the liquid medium for 3 days at 30.degree. C. using a shaking
culture method thereby obtaining a fermented supernatant, the fermented
supernatant is purified with ammonia sulfate precipitation thereby
obtaining the supernatant.

7. A method of producing nattokinases, comprising: fermenting a solution
of aquatic products castoff powder containing chitin using a Pseudomonas
sp. strain TKU015, which is deposited under DSMZ GmbH (Deutsche Sammlung
von Mikroorganismen and Zellkulturen GmbH) Number DSM 21747), thereby
obtaining a supernatant; and separating nattokinases from the
supernatant.

9. The method of producing nattokinases as claimed in claim 7, wherein a
concentration of the solution is in a range from 0.1% to 3%.

10. The method of producing nattokinases as claimed in claim 7, wherein
the solution comprises 0.2-1.5% by weight of shrimp shell powder (SSP),
0.05-0.2% by weight of K2HPO4, and 0.02-0.1% by weight of
MgSO.sub.4.7H2O, the Pseudomonas sp. strain TKU015 is cultured in
the solution using a shaking culture method.

11. The method of producing nattokinases as claimed in claim 10, wherein
the solution comprises 1% by weight of shrimp shell powder (SSP), 0.1% by
weight of K2HPO4, and 0.05% by weight of MgSO.sub.4.7H2O,
a volume of the solution is 100 mL, pH value of the solution is 7, the
Pseudomonas sp. strain TKU015 is cultured in the solution for 2 days at
30.degree. C. using a shaking culture method.

12. The method of producing nattokinases as claimed in claim 7, wherein
the separating step comprises: a. preparation of crude enzyme extract:
the supernatant is processed with ammonia sulfate precipitation and
centrifugal separation, remained sediments are re-dissolved with a few
phosphate buffer solution thereby obtaining a crude enzyme extract; b.
anion exchange chromatography: the crude enzyme extract is injected into
a column for performing column chromatography, non-adsorbed protein is
collected and then the column is eluted with phosphate buffer solution
containing NaCl at a gradient of from 1 to 0 M NaCl such that adsorbed
protein is eluted out, non-adsorbed area is collected as nattokinase
solution; and c. hydrophobic chromatography: nattokinase solution is
injected into a column and the column is eluted with phosphate buffer
solution containing ammonium sulfate, hydrophobicity of the column is
gradually changed such that nattokinases can be separated due to
different hydrophobicity.

Description:

REFERENCE TO SEQUENCE LISTING

[0001] A sequence listing is enclosed as an attachment, and the content of
the sequence listing information recorded in computer readable form is
identical to the written sequence listing.

BACKGROUND

[0002] Soybeans don't contain plasmin. Generally, natto is produced by
inoculating Bacillus natto or Bacillus subtilis into braised soybeans and
then fermenting the soybeans. A kinase, which names as nattokinase,
exists in viscousness distributed on surface of produced natto.
Nattokinase acts as a fibrinolytic enzyme of thrombus; therefore,
nattokinase is very useful in prevention and curing of brain stroke and
myocardial infarction, as well as Alzheimer's disease caused by
infarction in small vein.

[0003] Plasmin is the only fibrinolytic enzyme contained in human body.
Usually, blood contains a plasmin precursor called plasminogen.
Fibrinolytic enzyme is a plasminogen activator that can activate
plasminogen and thereby producing plasmin. Nattokinase has similar
properties of fibrinolytic enzyme; therefore, nattokinase is capable of
dissolving fibrin in blood serum. Furthermore, nattokinase reacts with
pro-urokinase together with a pro-urokinase activator thereby obtaining
urokinase. Urokinase reacts with plasminogen and fibrinolytic enzyme is
obtained. The obtained fibrinolytic enzyme further dissolves thrombus and
produces thrombus degradation products. In summary, nattokinase can
decreases thrombus by increasing amount of fibrinolytic enzyme.

[0004] Except in fermented soybean, nattokinase is also found in bacillus,
actinomyces, epiphyte and alga. However, there isn't any prior art
discloses that nattokinase can be produced from bacteria doesn't belong
to pseudomonas sp. and bacillus sp.

[0005] Shrimp and crab shell powder (SCSP) contains mass amount of protein
and chitin, if SCSP can be fermented with a microorganism to produce
valuable bioactive substance, pollution to the environment can be reduced
and value in use of SCSP can be improved.

BRIEF SUMMARY

[0006] In one exemplary embodiment, a Pseudomonas sp. strain TKU015 (gene
pool number EU103629, DSMZ GmbH (Deutsche Sammlung von Mikroorganismen
and Zellkulturen GmbH) Number DSM 21747) is provided as attachment A. The
Pseudomonas sp. strain TKU015 can be used to produce chitinase,
chitosanase and nattokinases.

[0007] In another exemplary embodiment, a method of producing nattokinase
is provided. The method utilizes the Pseudomonas sp. strain TKU015 to
ferment a solution of aquatic products castoff powder and nattokinases is
thereby obtained. Pollution of the aquatic products to environment is
reduced and value of the aquatic products is improved.

[0008] The strain TKU015 is selected from soil in North Taiwan and
cultured using shrimp shell powder as unique carbon/nitrogen source.
Identification results show that TKU015 belongs to Pseudomonas sp.

[0009] Nattokinase can be produced by fermenting a solution of aquatic
products castoff powder using the Pseudomonas sp. strain TKU015. The
aquatic products castoff comprises shrimp and crab shell, squid pen
power, mushroom and rude chitin. A concentration range of the aquatic
products castoff powder is i 0.1%-3%, preferably, the range is 0.2-1.5%.

[0011] Generally, culture medium that is suitable for producing chitinase
and chitosanase includes 0.2-1.5% by weight of shrimp shell powder (SSP),
0.05-0.2% by weight of K2HPO4, and 0.02-0.1% by weight of
MgSO4.7H2O. Preferably, the culture medium includes 1% by
weight of shrimp shell powder (SSP), 0.1% by weight of K2HPO4,
and 0.05% by weight of MgSO4.7H2O. The Pseudomonas sp. strain
TKU015 is cultured in the medium (pH 7) for 2 days at 30° C. The
obtained fermented supernatant is processed with Ammonium sulfate
precipitation, DEAE-Sepharose chromatography, and Phenyl-Sepharose
chromatography, and then nattokinase is separated. SDS-PAGE testing
results show that molecular weight of nattokinase is 21 kDa and 30 kDa
respectively. Optimum reacting temperature, pH stable range, thermal
stable range of nattokinase are pH 7, 50° C., pH 4-11, and
<37° C. Activity of nattokinase is completely inhibited by
PMSF. Fe2+ can improve activity of nattokinases.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] These and other features and advantages of the various embodiments
disclosed herein will be better understood with respect to the following
description and drawings, in which like numbers refer to like parts
throughout, and in which:

[0013]FIG. 1 is a microscope photograph of a Pseudomonas sp. strain
TKU015 provided in an embodiment of the present invention;

[0015]FIG. 3 is a graph showing influence of concentration of SSP to
produced nattokinases activity;

[0016]FIG. 4 is a graph showing influence of temperature to produced
nattokinases activity;

[0017]FIG. 5 is a graph showing influence of pH value to produced
nattokinases activity;

[0018]FIG. 6 is a graph showing influence of volume of culture medium to
produced nattokinases activity;

[0019]FIG. 7 is a graph showing growth curves of chitinase, chitosanase
and nattokinases produced in a culture medium of Pseudomonas sp. strain
TKU015; and

[0020]FIG. 8 is SDS-PAGE analysis graph of chitosanase and nattokinases
produced by Pseudomonas sp. strain TKU015.

DETAILED DESCRIPTION

[0021] Culture of New Strain TKU015

[0022] An amount of pre-gathered soil is dissolved in an appropriate
amount of germfree water and the obtained solution is coated on a solid
culture medium comprising 1% by weight of shrimp shell powder (SSP), 0.1%
by weight of K2HPO4, 0.05% by weight of MgSO4.7H20
and 1.5% by weight of agar. The medium is cultured for two days at
30° C., and the colony is observed. Produced single colony is
inoculated into a nutrient agar (NA) medium and cultured for one day at
30° C. After that, the NA medium is stored at 4° C. Single
colony filtrated from the NA medium is cultured in 100 mL liquid medium
disposed in a 250 mL conical flask for two days at 30° C. using a
shaking culture method. The liquid medium includes 1% by weight of SSP,
0.1% by weight of K2HPO4, and 0.05% by weight of
MgSO4.7H2O disposed. Finally, the liquid medium is
centrifugally purified, supernatant is separated, and activity of
chitinase and chitosanase of the supernatant is tested.

[0023] Characteristics of TKU015 Strain

[0024] As shown in FIG. 1, TKU015 is one kind of Gram-negative, which has
catalase, oxidase and motility, and can grow both in an aerobic and an
anaerobic environment, but does not produce spores.

[0025]FIG. 2 illustrates a portion of 16 S rDNA nucleotide sequence
analysis result, which indicates that TKU015 is most close to Pseudomonas
sp. and similarity of TKU015 and Pseudomonas sp. exceeds 97%.

[0026] Testing result of glucose metabolism type indicates that TKU015 is
oxidized type and is consistent with characteristics of Pseudomonas sp.

[0027] Analysis of fatty acid composition indicates that main fatty acid
composition of TKU015 includes C16:0 and H18:1ω7c, hydroxylated
fatty acids C10:0 3OH, C12:0 2OH and C12:0 3OH can also be found in
TKU015, but TKU015 doesn't contain C16:0 2OH and C16:0 3OH. The fatty
acid composition is also consistent with Pseudomonas sp.

[0028] Preparation of Chitin

[0029] In step a, squid pen powder (SPP) is mixed with a 2N NaOH solution
in a ratio of 3:40 (w/v), the obtained mixture is heated in a 100°
C. hot water bath for 30 minutes, residual and filtrate are separated by
filtrating, the residual is dried at 60° C. and then weighted. The
dried residual is mixed with a 2N HCl solution in a ration of 1:8 (w/v).
The residual is soaked fort two days at room temperature. Residual and
filtrate are separated by filtrating, the residual is dried at 60°
C. and then weighted.

[0030] In step b, 5 g residual is added into 50 mL of 12N HCl solution and
then stirred for 1 hour, the obtained mixture is poured into distilled
water, supernatant liquor is removed precipitation, and the residual is
washed with 50 mL H3PO4 (pH 7), the washing step is repeated
until pH value of supernatant equals to 7. After the supernatant is
removed, the obtained sediment is chitin suspension, which can be stored
at 4° C. for testing its activity of chitinase.

[0031] In order to produce chitinase and chitosanase, generally, TKU015 is
cultured in a liquid shaking culture medium (pH 8) including 0.5% by
weight of SSP, 0.1% by weight of K2HPO4 and 0.05% by weight of
MgSO4.7H2O for 3 days at 30° C. The obtained fermented
supernatant is purified with ammonia sulfate precipitation,
DEAE-Sepharose chromatography, Phenyl-Sepharose chromatography, and
Sephacryl S-100 chromatography in sequence, and a chitinase and a
chitosanase is obtained. Testing result of Sodium Dodecyl
Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) indicates that the
chitinase has a molecular weight of 68 kDa and the chitosanase has a
molecular weight of 30 kDa. An optimum pH value and an optimum
temperature for producing chitinase are pH 5 and 50° C., and
chitinase is stable under pH 5-7 and a temperature less than 60°
C. An optimum pH value and an optimum temperature for producing
chitosanase are pH 4 and 50° C., and chitinase is stable under pH
3-9 and a temperature less than 50° C. Activity of chitinase can
be prohibited by Mn2+ and Fe2+, and activity of chitosanase can
be inhibited by Mn2+, Cu2+, and phenylmethanesulfonyl fluoride
(PMSF).

[0032] Activity Test of Nattokinase

[0033] Protease activity of the supernatant obtained from culture medium
of chitinase (cultured in a liquid shaking culture medium (pH 8)
including 0.5% by weight of SSP, 0.1% by weight of K2HPO4 and
0.05% by weight of MgSO4.7H2O for 3 days at 30° C.) is
respectively tested using casein and N-succinyl-Ala-Ala-Pro-Phe-pNa as
substrate. The supernatant hasn't protease activity when casein is
employed as substrate. In contrast, the supernatant has protease activity
when N-succinyl-Ala-Ala-Pro-Phe-pNa is employed as substrate; this
indicates that the supernatant has nattokinase activity (0.02 FU/mL). In
summary, the supernatant contains chitinase, chitosanase and nattokinase.

[0034] Selection of Carbon/Nitrogen Source

[0035] Chitosanase and nattokinase also exist in supernatant obtained from
culture medium that is cultured under an appropriate environment for
producing chitinase. In order to maximize output of nattokinase, SSP is
employed as main carbon source of nattokinase. In addition, other
condition of producing nattokinase is also discussed. Preferably, a
concentration of SSP is 1% by weigh (3.8 FU/mL) (referring to FIG. 3).

[0036] Appropriate Condition for Producing Nattokinase

[0037] As described above, appropriate carbon source for producing
nattokinase is 1% SSP. The carbon source is cultured under different
temperature (25° C., 30° C., 37° C.) for 1-4 days.
The nattokinase activity testing results are shown in FIG. 4, when
cultured for 2 days at 30° C., a highest nattokinase activity (4
FU/mL) is obtained. 30° C. is most appropriate temperature for
producing nattokinase.

[0038] Culture mediums have different pH values (5-10) is cultured for 1-4
days at 30° C., and nattokinase activity testing results are shown
in FIG. 5, when the pH value of culture medium is 7 a highest nattokinase
activity (4 FU/mL) is obtained.

[0039] Different volumes (50 mL, 100 mL, 150 mL, and 200 mL) of culture
medium, which contains 1% SSP and has a pH value of 8, is disposed in a
250 conical flask and cultured for 1-4 days at 30° C. nattokinase
activity testing results are shown in FIG. 6, when the volume of culture
medium is 100 mL a highest nattokinase activity (5.5 FU/mL) is obtained.

[0040] In summary, optimum conditions for producing nattokinase include:
100 mL shaking culture medium (pH 7) including 1% by weight of SSP, 0.1%
by weight of K2PO4 and 0.05% by weight of MgSO4.7H2O
cultured for 2 days at 30° C., which can result in a highest
nattokinase activity.

[0041]FIG. 7 illustrates a growth curve of Pseudomonas sp. TKU015
cultured in a shaking liquid medium (pH 8) including 0.5% by weight of
SSP, 0.1% by weight of K2HPO4, and 0.05% by weight of
MgSO4.7H2O at 30° C. It is known that nattokinase activity is
highest on the second day, and then nattokinase activity decreases with
increasing of time. The chitinase and chitosanase activity is highest on
the third day.

[0042] Separating and Purifying of Nattokinase

[0043] Crude enzyme extract preparation: TKU015 is cultured in an
appropriate culture medium; the obtained fermentation broth is processed
with ammonium sulfate precipitation and centrifugal separation; remained
sediments are re-dissolved with a few 50 mM phosphate buffer solution (pH
7) and then dialyze at 4° C.; the resulted dialysis fluid is crude
enzyme extract (50 mL).

[0044] Anion exchange chromatography: the crude enzyme extract is injected
into a column for performing DEAE-Sepharose CL-6B column chromatography,
a flow rate of the crude enzyme extract is 4 mL/min, every 6 mL of
resulted solution is collected in a cuvette, non-adsorbed protein is
collected, and then the column is eluted with 50 mM phosphate buffer
solution (pH 7) that contain NaCl at a gradient of 0-1 M such that
adsorbed protein is eluted out; nattokinase is found in non-adsorbed area
and is collected as nattokinase solution (100 mL).

[0045] Hydrophobic chromatography: 80 mL of nattokinase solution
(containing 1 M ammonium sulfate) is injected into a Phenyl Sepharose 6
Fast Flow column, a flow rate of the nattokinase solution is 4 mL/min,
each 6 mL of resulted solution is collected in a cuvette; firstly, the
column is eluted with 50 mM phosphate buffer solution (pH 7) that contain
1 M ammonium sulfate; and then the column is eluted with different 50 mM
phosphate buffer solution (pH 7) that contain from 1 M to 0 M of NaCl to
change hydrophobicity of the column, the nattokinase solution can be
separated under at different hydrophobicity. Finally, the column is
eluted with 50 mM phosphate buffer solution (pH 7) to wash hydrophobic
protein out of the column. Area having nattokinase activity is collected
(70 mL) and enzyme characteristics analysis and SDS-PAGE is performed.

[0046] Comprehensive Results: during the purification process, nattokinase
exists in non-adsorbed area of the DEAE-Sepharose column, after the
followed hydrophobic Phenyl-Sepharose chromatography step, TKU015
nattokinase (45 mg) can be separated. DEAE-Sepharose separation results
at pH 7 show that chitosan and nattokinase both exists in non-adsorbed
area and isoelectric points of both should belong to alkaline pH.

[0047] DEAE-Sepharose separation results show that chitosan appears after
the solvent gradient is increased; therefore, chitosan should be a
hydrophobic protein. In contrast, nattokinase appears prior to the
solvent gradient is increased; therefore, nattokinase should be a
hydrophilic protein. Purification ration of nattokinase is 5.1.

[0048] The Molecular Weight Determination of Nattokinase

[0049] SDS-PAGE: molecular weight of nattokinase is analyzed using 12.5%
SDS-PAGE, and testing result is shown in FIG. 8, in which Lane 1 belongs
to crude enzyme extract and Lane 4 belongs to TKU015 nattokinase.
Molecular weight of nattokinase is about 21 kDa.

[0050] Comprehensive Results: molecular weight of TKU015 nattokinase is
about 21 kDa, as shown in Table 1, molecular weight of majority of
nattokinase that are produced from microorganisms are in a range from 20
kDa to 45 kDa. Currently, it is not found that Pseudomonas is used in
production of nattokinase. Molecular weight of TKU015 nattokinase is less
than that of nattokinase produced by bacteria, and is similar to that of
nattokinase produced by fungi such as Armillaria mellea AMMP (21 kDa) and
Rhizopus chinensis. 12(18 kDa). As shown in Table 1, majority of
nattokinase are produced by bacteria and fungi. Molecular weight of
nattokinase produced from bacteria is about 30 kDa, and nattokinase
produced from the Pseudomonas sp. TKU015 is similar to that of fungi.

[0052] Optimum temperature and thermal stability range of TKU015
nattokinase are 50° C. and <37° C. As shown in table 1,
most optimum temperature and thermal stability range of nattokinase are
37-50° C. and <50° C. With respect to optimum
temperature, nattokinases produced by bacteria of Bacillus subtilis KCK-7
(50° C., Bacillus subtilis Strain A1 (50° C.) and Bacillus
amyloliquefaciens DC-4 (48° C.) are similar to TKU015 nattokinase.
With respect to thermal stability range, TKU015 nattokinase is relatively
non-stable to heat, nattokinase produced by fungi such as Rhizopus
chinensis. 12 (<37° C.), Fomitella fraxinea FFP2
(<40° C.), (Park et al, 2005) is similar to TKU015 nattokinase.
Above results indicate that molecular weight and temperature
characteristics of Pseudomonas sp. TKU015 nattokinase are similar to that
of fungi nattokinase.

[0053] Optimum pH value and pH stability range of TKU015 nattokinase are
pH 7 and pH 4-11. As shown in Table 1, most optimum pH values of
nattokinase are in a range from neutral to a little alkaline, and pH
stability range are pH 7-10. With respect to optimum pH value,
nattokinases produced by Bacillus sp. KA38 (pH 7), Bacillus sp. KDO-13
(pH 7), and Bacillus subtilis Strain A1 (pH 7) are similar to TKU015
nattokinase. With respect to pH stability range, TKU015 nattokinase is
stable in a weakly acidic or a weakly alkaline environment, and can
remain 80% of activity in a solution having a pH value of 11,
nattokinases produced by Bacillus amyloliquefaciens DC-4 (pH 4-11) and
Bacillus sp. subtilisin QK-2 (pH 3-12) are similar to TKU015 nattokinase.

[0056] Table 4 shows influence of different organic solvents on TKU015
nattokinase activity. TKU015 nattokinase activity increase to 150% in the
presence of acetone; methanol, toluene and xylene do not affect TKU015
nattokinase activity; ether inhibits 50% of TKU015 nattokinase activity;
and TKU015 nattokinase remain about 70% of activity in the other
solvents. With respect to Pseudomonas protease, activity of Pseudomonas
aeruginosa PseA, (Khare et al., 2005) doesn't change in the presence of
toluene and N,N-dimethylformamide (DMF); activity of Pseudomonas
aeruginosa PST-01, (Ogino et al., 1999) doesn't change in the presence of
toluene, DMF and actone; activity of Pseudomonas aeruginosa san-ai
strain, (Lee et al., 2006) doesn't change in the presence of DMF. Similar
to PseA protease, PST-01 protease and san-ai protease, TKU015 nattokinase
remains 80% of activity in the presence of DMF. In addition, activity of
TKU015 nattokinase and PST-01 protease respectively increased to 150% and
200% in the presence of acetone. With respect to thermal stability ,
TKU015 nattokinase is mixed with different solvents and then respectively
placed for ten days at 25° C. and 4° C. As shown in Table
5, the solvents don't affect activity of nattokinase at 25° C. and
4° C. Similarly, the same effects are also found in Pseudomonas
sp. PseA protease (Khare et al., 2005) and Pseudomonas san-ai protease
(Lee et al., 2006).

[0057] Pseudomonas sp. TKU015 is cultured in a liquid medium (100 mL/250
mL), which employ shrimp shell powder as carbon/nitrogen source, for 2-3
days at 30° C., and chitinase, chitosanase and nattokinase
activity are tested in the medium. The fermented supernatant is processed
by Ammonium sulfate precipitation, DEAE-Sepharose chromatography,
Phenyl-Sepharose chromatography and Sephacryl S-100 chromatography, and
then three pure enzymes (chitinase, chitosanase and nattokinase) are
obtained. Test results of SDS-PAGE show molecular weight thereof are
respectively 68 kDa, 30 kDa and 21 kDa. Respectively, optimum reacting pH
value, optimum reacting temperature, pH stable range, thermal stable
range of chitinase are pH 5, 50° C., pH 5-7, and <60°
C.; optimum reacting pH value, optimum reacting temperature, pH stable
range, thermal stable range of chitosanase are pH 4, 50° C., pH
3-9, and <50° C.; and optimum reacting pH value, optimum
reacting temperature, pH stable range, thermal stable range of
nattokinase are pH 7, 50° C., pH 4-11, and <37° C.
Activity of chitinase is inhibited by Mn2+ and Fe2+, activity
of chitosanase is inhibited by Mn2+, Cu2+ and PMSF, and
activity of nattokinase is completely inhibited by PMSF. Nattokinase
belongs to serine-type protease, and Fe2+ can increase its activity.
Acetone can affect activity of chitinase, acetonitrile can affect
activity of chitosanase, and ether can affect activity of nattokinase.
With respect to organic solvent resistance, after placed for 10 days at
25° C. and 4° C., test results show that toluene, xylene
and DMF don't affect activity of chitinase while the other organic
solvents decrease activity of chitinase at 25° C., ether,
acetonitrile and acetone can affect activity of chitinase; toluene,
xylene and DMF don't affect activity of chitosanase while the other
organic solvents decrease activity of chitosanase at 25° C., and
ether and acetone can affect activity of chitosanase at 4° C.; and
all the organic solvents don't affect activity of nattokinase.

[0058] Generally, shrimp and crab shell are useless and often thrown away.
However shrimp and crab shell contains mass amount of protein and chitin,
the present invention utilize a new strain TKU015 to ferment the shrimp
and crab shell, and finally, valuable bioactive substance nattokinase can
be produced, pollution to environment is reduced and value in use of
shrimp shell castoff is improved.

[0059] The above description is given by way of example, and not
limitation. Given the above disclosure, one skilled in the art could
devise variations that are within the scope and spirit of the invention
disclosed herein, including configurations ways of the recessed portions
and materials and/or designs of the attaching structures. Further, the
various features of the embodiments disclosed herein can be used alone,
or in varying combinations with each other and are not intended to be
limited to the specific combination described herein. Thus, the scope of
the claims is not to be limited by the illustrated embodiments.